The goal of this proposal is to test, and equally importantly rationalize, the cellular activity and specificity of anti-HIV lead compounds that targt the transactivation response element (TAR) RNA in the HIV LTR. The leads are identified in related studies by the two principal investigators using a new 'dynamics-based' virtual screen. These studies establish the binding and activity characteristic of lead compounds in vitro with unprecedented depth and breadth using an assortment of biophysical techniques, but they do not include the key biological assays needed to identify promising leads that could be developed further into anti-HIV therapeutics. By subjecting a strategically chosen panel of these 'highly biophysically characterized' lead compounds to rigorous toxicity as well as biological assays against HIV-1 and the closely related HIV-2, we propose to (i) identify promising leads with in vivo activity that may be subjected to further optimization in the developed of anti-HIV therapeutics and (ii) delineate the link between the biophysical properties of RNA-small molecule binding, which can be obtained in a semi- high throughput manner in vitro, and biological activity and specificity, which is far more cumbersome to experimentally assay. We will test the hypothesis that 'RNA-targeting specificity' is a major determinant of cellular activiy and toxicity and the main reason RNA-targeting compounds with in vitro activity fail to exhibit in vivo anti-viral activity. We will develop in vitro assays for quantifying RNA-targeting specificity and use a new specificity metric to narrow down compounds that are subjected to cellular assays as well as test a new strategy for targeting flexible RNA systems with exquisite selectivity that is based on regiospecifically crowding cationic groups. By bridging the divide between in-depth biophysical properties and biological assays, our studies will lay the foundations for a predictive understanding of RNA- targeting that can be used to rationally identify compounds with anti-retroviral activity.